The biosynthetic and respiratory capacities of mitochondria must adapt to cellular demand and environmental conditions to ensure organismal health, but control mechanisms for these adaptations are little understood. The protein unfoldase ClpX is an important regulatory element in prokaryotes, mediating changes in cell state and responses to stress conditions through its selection of substrates. In eukaryotes, ClpX is a widely conserved component of the mitochondrion, but no mitochondrial ClpX substrates have yet been discovered, and the specific contributions of ClpX to mitochondrial physiology are not known. This proposal seeks to define how mitochondrial ClpX modulates mitochondrial physiology. Analysis of large-scale genetic interaction maps in S. cerevisiae and phenotypic assays indicated that the yeast mitochondrial ClpX homolog, Mcx1, promotes the first step in the biosynthesis of heme. Metabolic profiling will be used to define the contribution of Mcx1 to this step and to narrow a set of candidate substrates. The identity and mechanism of the substrate interaction through which Mcx1 modulates heme biosynthesis will be determined through complementary in vivo and in vitro assays for substrate processing by Mcx1. These efforts will define a novel mechanism by which the biosynthesis of an essential cofactor is regulated. Phenotypic data in S. cerevisiae and C. elegans, as well as analogy with its prokaryotic homologs, indicate that mitochondrial ClpX may regulate other mitochondrial processes as well. Through an activity-based protein trapping strategy combined with mass spectrometry, the potentially broader repertoire of physiological substrates of Mcx1 will be sampled; this strategy may indicate other mitochondrial processes that Mcx1 regulates, and will allow common motifs that target mitochondrial proteins to Mcx1 to be defined. These studies seek to define the functional repertoire of a mitochondrial regulator, the ClpX homolog Mcx1, and the mechanisms by which its activity is controlled, through a targeted approach toward understanding its role as a control element in the biosynthesis of the essential cofactor heme, and through an unbiased approach that will sample the broader contribution of Mcx1 to mitochondrial physiology. These studies could inform the development of new targets for therapy in anemias resulting from aberrant heme biosynthesis as well as porphyrias. In addition, mitochondrial maladaptation underlies many other diseases in humans, including metabolic diseases and various degenerative ailments resulting from diabetes. Defining molecular mechanisms by which mitochondrial respond and adapt to cellular demand and to environmental stress will provide a new framework for understanding how failures in these responses contribute to disease.